An association test of the spatial distribution of rare missense variants within protein structures identifies Alzheimer's disease–related patterns

More than 90% of genetic variants are rare in most modern sequencing studies, such as the Alzheimer''s Disease Sequencing Project (ADSP) whole-exome sequencing (WES) data. Furthermore, 54% of the rare variants in ADSP WES are singletons. However, both single variant and unit-based tests are limited in their statistical power to detect an association between rare variants and phenotypes. To best use missense rare variants and investigate their biological effect, we examine their association with phenotypes in the context of protein structures. We developed a protein structure–based approach, protein optimized kernel evaluation of missense nucleotides (POKEMON), which evaluates rare missense variants based on their spatial distribution within a protein rather than their allele frequency. The hypothesis behind this test is that the three-dimensional spatial distribution of variants within a protein structure provides functional context to power an association test. POKEMON identified three candidate genes (TREM2, SORL1, and EXOC3L4) and another suggestive gene from the ADSP WES data. For TREM2 and SORL1, two known Alzheimer''s disease (AD) genes, the signal from the spatial cluster is stable even if we exclude known AD risk variants, indicating the presence of additional low-frequency risk variants within these genes. EXOC3L4 is a novel AD risk gene that has a cluster of variants primarily shared by case subjects around the Sec6 domain. This cluster is also validated in an independent replication data set and a validation data set with a larger sample size.

High-throughput DNA sequencing of diverse humans has identified millions of genetic variants, the vast majority of which are exceptionally rare. A survey of ∼60,000 individuals from the Exome Aggregation Consortium (ExAC) found that out of ∼7 million variants, 99% have a frequency <1% and 54% are singletons (Taliun et al. 2021). Similarly, in the Alzheimer''s Disease Sequencing Project (ADSP) whole-exome sequencing (WES) of ∼10,000 individuals, 97% of identified variants have a minor allele frequency <1%, and 23% are singletons (Butkiewicz et al. 2018). However, the effect of most rare variants on diseases of interest remains unknown because of insufficient statistical power to detect the associations between these variants and phenotypes.We hypothesized that rare missense variants contribute to common diseases by disrupting the protein function and are likely to form clustered or dispersed patterns within protein structures when examined in population-based studies. Therefore, incorporating spatial context will improve rare variant association tests. Prior studies have shown that missense variants show nonrandom patterns in protein structures, such as cancer-associated hotspot regions with a high density of missense somatic mutations (Tokheim et al. 2016). Our group (Sivley et al. 2018) also found that germline causal missense variants for Mendelian diseases show nonrandom patterns in three-dimensional (3D) space. These patterns include clusters that likely reflect disruption of a key functional region and dispersions that likely reflect depletion of variants within a sensitive protein core.To test this hypothesis within sequencing studies of disease traits, we developed a kernel function to quantify genetic similarity among individuals by using protein structure information. When two individuals have different missense variants distal in genomic coordinates but close in 3D protein structure, these individuals will be assigned a high genetic similarity through our kernel function. When applied over an entire data set, our kernel function captures differences in the spatial patterns of rare missense variants among cases and controls or over continuous traits. Using a statistical framework similar to SKAT (Wu et al. 2011), we test the association of rare variants with quantitative and dichotomous phenotypes using this structure-based kernel. We call this approach protein optimized kernel evaluation of missense nucleotides (POKEMON). We validated that POKEMON can identify trait associations with spatial patterns formed by missense variants both in simulation studies and real-world data.     

Anticonvulsant effect of celecoxib on pentylenetetrazole-induced convulsion: Modulation by NO pathway

This study aimed to examine whether celecoxib influences clonic seizure thresholds through modulation of nitric oxidergic (NO) pathway. The effect of celecoxib (1-5 mg per kg, p.o.) was investigated on clonic seizures induced by pentylenetetrazole (PTZ, 50 and 80 mg per kg, i.p.) in male Swiss mice. The interaction of celecoxib-induced effects with NO pathway was examined using a NO synthase (NOS) inhibitor, N(G)-omega-nitro-L-arginine methyl ester (L-NAME, 20 and 50 mg per kg, i.p.) and a NOS substrate, L-arginine (100 and 200 mg per kg, i.p.). The criteria for the development of seizure activity were the possibility for appearance of generalized clonus and prolongation of latency to the onset of convulsions following administration of 50 and 80 mg per kg of PTZ, respectively. Pretreatment with celecoxib (2.5 and 5 mg per kg) or L-NAME (50 mg per kg) induced anticonvulsant effect on the PTZ-induced clonic seizures. L-arginine at the dose of 200 mg per kg had proconvulsant effect. A sub-effective dose of celecoxib (1 mg per kg) induced an additive anticonvulsant effect when co-administered with L-NAME (20 mg per kg). Although L-arginine (100 mg per kg) per se did not influence PTZ-induced convulsion, it could attenuate the anticonvulsant effect of celecoxib (5 mg per kg). Our results indicate that celecoxib induces an anticonvulsant effect on clonic seizure threshold that may involve NO pathway.     

Potential effect of opium addiction on lipid profile and blood glucose concentration in type 2 diabetic patients in Iran

Background: Diabetes is one of the most common chronic diseases in the world. People believe that opium improves blood glucose and lipid profiles in these patients and controversial studies show the effect of consumption of opium in controlling these indices. Accordingly, this study aimed to compare the serum levels of blood indices such as fasting blood glucose (FBS), Hemoglobin A1c (HbA1c) and lipid profile in opium users and non-users in type ΙΙ diabetic patients.

Methods: In this cross-sectional study, among type II diabetic patients referred to the Diabetes Clinic of Birjand 45 opium users and 135 non-users were selected and entered the study by the convenience sampling method.

Results: The results of this study showed that the mean serum levels of FBS, HbA1c, and serum lipid profiles were not significantly different between the two groups of opium users and non-users, but the levels of triglyceride (TG) were significantly 0.18 unit higher in the opium users, compared to non-opium users.

Conclusion: According to the results, the use of opium does not affect the indices of blood glucose, HbA1c and serum lipid profiles except triglyceride in diabetic patients. The general belief that opioid use reduces biochemical indices does not seem to be correct.

Abbreviations: FBS: fasting blood sugar; HbA1c: Hemoglobin A1c; LDL: low-density lipoprotein; HDL: High-density lipoproteins; HDL-C: High-density lipoproteins-cholesterol; BMI: Body mass index; IQR: Inter quartile range; TG: triglyceride; TC: total cholesterol; NS: non-significant; S: significant; ATN: Acute tubular necrosis     

Pharmacophore-based predictive model generation for potent antimalarials targeting haem detoxification pathway

Pharmacophore hypotheses were developed for molecules having antimalarial activities targeting the haem detoxification pathway of the malaria parasite. A training set consisting of 23 compounds was selected to generate these hypotheses, and their activities were evaluated for haem polymerization inhibition and against chloroquine-sensitive (3D7) as well as chloroquine-resistant (K1) strains of p. falciparum. The models were cross-validated by Fischer’s randomization test at a 95% confidence level. The model developed against chloroquine-resistant malaria parasites was found to yield the best predictions among the three models.     

Fluorescent zinc indicators for neurobiology

Mounting evidence indicates that zinc has multiple roles in cell biology, viz. as a part of metalloenzyme catalytic sites, as a structural component of gene regulatory proteins, and (like calcium) as a free signal ion, particularly in the cortex of the brain. While most Zn(II) in the brain is tightly bound, such that free Zn(II) levels extracellularly and intracellularly are likely to be picomolar, a subset of glutamatergic neurons possess weakly bound zinc in presynaptic boutons which is released at micromolar levels in response to a variety of stimuli. Key to further progress in understanding the multiple roles of zinc will be the availability of fluorescent indicator systems that will permit quantitative determination and imaging of zinc fluxes and levels over a broad concentration range both intracellularly and extracellularly using fluorescence microscopy. Towards that end, we have compared a variety of fluorescent indicators for their sensitivity to Zn(II) and Cu(II), selectivity for Zn(II) in the presence of potential interferents such as Ca(II) or Mg(II), and potential for quantitative imaging. The commercially available probes Fura-2, Mag-Fura-5, Newport Green DCF, and FuraZin-1 were compared with the carbonic anhydrase-based indicator systems for selectivity and sensitivity. In addition, intracellular levels of Zn following excitotoxic insult were determined by single pixel fluorescence lifetime microscopy of Newport Green DCF, and extracellular levels of free zinc following stimulus of rat hippocampal slices were determined ratiometrically with a carbonic anhydrase-based indicator system. These results suggest that zinc ion at high nM to microM levels can be accurately quantitated by FuraZin-1 ratiometrically or by Newport Green DCF by fluorescence lifetime; and at levels down to pM by intensity ratio, lifetime, or polarization using carbonic anhydrase-based systems.     

The fetal and neonatal brain protein neuronatin protects PC12 cells against certain types of toxic insult

The protein neuronatin is expressed in the nervous system of the fetus and neonate at a much higher level than in the adult. Its function is unknown. As a result of variable splicing, neuronatin mRNA exists in two forms, alpha and beta. Wild type PC12 cells express neuronatin-alpha. We have isolated a PC12 variant, called 1.9, that retains many of the neuron-like properties of wild type PC12 cells, but it does not express neuronatin and it exhibits markedly increased sensitivity to the toxic effects of nigericin, rotenone and valinomycin. Pretreatment of the 1.9 cells with alpha-methyltyrosine, which inhibits dopamine synthesis, had little effect on the cells' sensitivity to nigericin, rotenone or valinomycin indicating that dopamine-induced oxidative stress was not involved in the toxicity of these compounds. However, flattened cell subvariants of the 1.9 cells, which do not have any neuron-specific characteristics, did not exhibit increased sensitivity to nigericin indicating that some neuronal characteristic of the 1.9 cells contributed to the toxicity of nigericin. After the neuronatin-beta gene was transfected into and expressed in the 1.9 cells, they regained wild type PC12 levels of resistance to nigericin, rotenone and valinomycin. These studies suggest that the function of neuronatin during development could be to protect developing cells from toxic insult occurring during that period.